A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.


Introduction
Melanoma, arising from pigment producing melanocytes, is the deadliest form of skin cancer, with an estimated 100,640 new cases and 8,290 deaths in the United States in 2024 alone (Siegel et al., 2024).The most common mutation in melanoma is BRAF V600E , which accounts for approximately 50% of melanoma cases and results in activation of the MAPK pathway promoting cell growth and survival (Akbani et al., 2015;Lo and Fisher, 2014).In addition, developmental signaling pathways are commonly dysregulated.Melanoma cells have increased expression and secretion of TGFb ligands compared to normal melanocytes, and TGFb ligand expression correlates with melanoma progression (Van Belle et al., 1996;Albino et al., 1991;Rodeck et al., 1991;Rodeck et al., 1994;Krasagakis et al., 1998;Javelaud et al., 2008;Perrot et al., 2013;Rak et al., 1996;Cerami et al., 2012;Gao et al., 2013;Lauden et al., 2014;Schmid et al., 1995).TGFb ligand binding to receptors on the cell surface results in phosphorylation and activation of SMAD2 and SMAD3 transcription factors.SMAD2 and SMAD3 translocate to the nucleus with SMAD4 to modulate gene expression (Batlle and Massagué, 2019).In normal melanocytes and early melanoma, TGFb acts as a tumor suppressor.However, in advanced melanoma TGFb is pro-tumorigenic as it induces growth, invasion, and metastasis (Perrot et al., 2013).As current targeted MAPK and immune checkpoint inhibitors often result in resistance, there is a need to study additional pathways perturbed in melanoma, such as TGFb.
Most cells in the tumor microenvironment can respond to and initiate TGFb signaling, although this often occurs in a heterogenous manner (Derynck et al., 2021).Generally, TGFb has an immunosuppressive effect in advanced tumors, resulting in inactivation of cytotoxic CD8 + T cells, expansion of immune suppressive regulatory T cells, inhibition of dendritic cell antigen presentation, and conversion of macrophages to an anti-inflammatory and pro-angiogenic M2-like state (Batlle and Massagué, 2019;Derynck et al., 2021;Naganuma et al., 1996;Ahmadzadeh and Rosenberg, 2005;Donkor et al., 2011;Fridlender et al., 2009;Mantovani et al., 2002;Standiford et al., 2011;Kobie et al., 2003).TGFb can act as a chemoattractant for macrophages and monocytes to areas of inflammation.Recruitment of monocytes by TGFb results in differentiation into macrophages that attach to the extracellular matrix (ECM) or promote blood vessel leakiness allowing for tumor cell extravasation (Arwert et al., 2018;Kelly et al., 2018;Kim et al., 2021;Allen et al., 1990;Wahl et al., 1993).In colorectal and urothelial cancers, TGFb in the tumor microenvironment was found to mediate immune evasion such that TGFb inhibition rendered these tumors susceptible to anti-PD-1/PD-L1 immune checkpoint inhibitors (Tauriello et al., 2018;Mariathasan et al., 2018).Due to its immunosuppressive effect, several TGFb inhibitors are in clinical trials in combination with immune checkpoint inhibitors (Lan et al., 2018;Paz-Ares et al., 2020;Strauss et al., 2018;Martin et al., 2020;Welsh et al., 2021).
Here, we visualized TGFb response across zebrafish melanoma tumorigenesis.A human melanoma enhancer, induced upon TGFB1 signaling was identified, and stable transgenic zebrafish with this enhancer driving EGFP were generated (TIE:EGFP).This TGFb inducible enhancer reporter is expressed in spatially distinct regions in advanced zebrafish melanomas and is characterized by up-regulation of a series of novel chronic TGFb target genes involved in the extracellular matrix.Single-cell RNA-seq and confocal microscopy revealed that TIE:EGFP + melanoma cells down-regulate interferon response and are preferentially phagocytosed by macrophages.Overexpression of the chromatin remodeler SATB2, which is associated with tumor spreading, shows early activation of TGFb signaling in these melanomas, suggesting that specific melanoma genotypes may benefit from TGFb inhibition.Overall, this work demonstrates the need for biomarker development to predict response to TGFb inhibitors in advanced or aggressive melanoma subtypes.

A TGFb enhancer reporter is inducible and specific in zebrafish
To visualize dynamic TGFb response across melanoma development, we designed a TGFb Inducible Enhancer (TIE) reporter using human melanoma cell (A375) ChIP-seq data following a 2-hr TGFB1 treatment.RNA-sequencing indicated that following a 2-hr treatment of A375 cells with 10 ng/mL human recombinant TGFB1, 223 genes were significantly up-regulated (q<0.05)including typical TGFb target genes SMAD7, JUNB, and PMEPA1, while 94 genes were down-regulated (Figure 1figure supplement 2A, left).Hallmark gene set enrichment analysis (GSEA) of genes ranked by log2 fold-change (log2fc) confirmed that TGFb was the top up-regulated pathway following 2-hr treatment (q=0) (Figure 1-figure supplement 2A, right).This indicates that a 2-hr treatment with human recombinant TGFB1 ligand is sufficient to activate the TGFb pathway.
To identify a TGFB1 inducible enhancer region, we selected a region of chromatin that was exclusively open upon stimulation, based on H3K27ac ChIP-seq, with unique SMAD2/3 binding following treatment (Figure 1A).The enhancer region under the SMAD2/3 peak was cloned upstream of a betaglobin minimal promoter and EGFP in a Tol2 vector backbone.TIE:EGFP was tested for inducibility in the presence of ubiquitously expressed constitutively active SMAD2 and SMAD3 (ubi:caSMAD2/3) by electroporation of adult zebrafish skin, with ubiquitous BFP (ubi:BFP) as a control for electroporation efficiency.TIE:EGFP reporter activity was significantly increased in the presence of ubi:caSMAD2 and ubi:caSMAD3, confirming that the reporter is inducible (Figure 1-figure supplement 1A).

TIE:EGFP reporter is expressed in advanced zebrafish melanomas
To visualize TGFb response across melanoma development, single cell Tg(mitfa:BRAF V600E );p53 -/-;mitfa -/- embryos stably expressing TIE:EGFP were injected with an empty MiniCoopR vector (MCR:MCS) containing the mitfa minigene to generate melanomas.Tyrosinase gRNA and Cas9 protein were also injected to knock-out melanocyte pigment as well as mitfa:mCherry to allow for melanocyte visualization.Normal melanocytes rarely had TIE:EGFP expression, with the exception of occasional fin melanocytes (Figure 1-figure supplement 1D).TIE:EGFP has yet to be observed in mitfa:mCherry high early melanomas (Figure 1-figure supplement 1E).However, of the fish with melanoma formation (n=56), 55% turned on TIE:EGFP in advanced melanomas defined by protrusion from the body plane (Figure 1C).TIE:EGFP + cells often occur in clusters throughout the tumor and these cells are TIE:EGFP + for many weeks at a time.This data indicates that advanced zebrafish melanomas develop TGFb responsive zones.

TIE:EGFP expressing melanoma cells down-regulate interferon signaling
To understand the transcriptional differences between TIE:EGFP + and TIE:EGFP -melanoma cells, single TIE:EGFP + and mitfa:mCherry + cells from TIE:EGFP expressing melanomas were processed for single cell RNA-sequencing at 23 and 42 weeks post fertilization (wpf), respectively (melanomas used are depicted in Figure 1C and Figure 1-figure supplement 2B).We used SORT-seq to sequence flow cytometry-sorted single cells and in silico linked transcriptomes to fluorescence intensities as recorded by FACS indexing (Muraro et al., 2016).A UMAP of the sequencing results from our combined replicates is shown (Figure 2A; UMAP with no batch correction in Figure 2-figure supplement 1A).Most cells were identified as melanoma cells expressing mitfa and/or sox10, but we identified an mpeg1.1expressingTIE:EGFP + macrophage cluster as well (Figure 2A).
The melanoma cell population was separated into TIE:EGFP -, TIE:EGFP low , and TIE:EGFP high based on FACS intensities.We found that immediate-early TGFb target genes, identified following an acute 2 hr TGFB1 treatment in zebrafish melanoma cells, ZMEL1, were down-regulated in TIE:EGFP high cells (Figure 2B).However, we identified 29 genes that were up-regulated in TIE:EGFP high cells.We termed these genes, related to the extracellular matrix, 'chronic' TGFb targets (Figure 2C; Katsuno et al., 2019;Verrecchia et al., 2001).At the time of dissociation, these melanomas had TIE:EGFP + cells for several weeks, therefore they were likely not in an acute TGFb response phase (Figure 1C and Figure 1-figure supplement 2B).Using GSEA analysis we found that the top down-regulated pathways in TIE:EGFP high cells were interferon alpha and gamma (Figure 2-figure supplement 1B).This suggests that TGFb has an immune suppressive effect in these melanomas.

AP-1 factors are required for induction of the chronic TIE reporter
Although the vast majority of literature focuses on acute TGFb signaling, our TIE:EGFP reporter remains on in melanoma, reading out chronic TGFb signal.We next asked what transcription factors are necessary for induction of our novel chronic TGFb reporter and therefore required for chronic TGFb signaling.We performed HOMER motif analysis of ~12,000 SMAD2/3 responsive regulatory elements.These regions were identified in A375 melanoma cells stimulated with TGFB1 and were bound by SMAD2/3 upon stimulus.We found that these SMAD2/3 bound regions are highly enriched for AP-1 motifs (Figure 2D).ChIP-seq in A375 cells in the presence or absence of TGFB1 treatment confirmed the binding of AP-1 transcription factors JUNB and ATF3 (Figure 2E).ATF3 and JUNB were bound at 19% and 48% of SMAD2/3 responsive elements, respectively, before TGFB1 treatment was administered.This data indicates that AP-1 factors, in particular JUNB, may be important for SMAD binding to chromatin.
We hypothesized that AP-1 factors occupy SMAD responsive elements prior to stimulation, stabilizing open chromatin to allow the TGFb response to occur quickly upon stimulation.To test this hypothesis, we deleted AP-1 motifs in our TIE enhancer driving luciferase.We identified AP-1 motifs (highlighted in orange) in our TGFb-induced enhancer using MoLoTool and HOCOMOCO motifs (Figure 2F, top; Kulakovskiy et al., 2018).AP-1 motif #1 was the most significant (p=8.4e - ), and AP-1 motif #2 had a lesser p-value of 8.2e -4 .We deleted either AP-1 site #1 alone, AP-1 site #2 alone, or both AP-1 sites, as well as the most significant SMAD2/3 motif (p=6.7e - ), shown in red (Figure 2figure supplement 2).In A375 cells, loss of AP-1 site #1 rendered the enhancer region unresponsive to TGFb signaling, however loss of AP-1 site #2 did not alter inducibility (Figure 2F,bottom).This result indicates that AP-1 site #1 is necessary for TGFb inducibility at this enhancer.Deletion of the SMAD site also destroyed TGFB1 responsiveness.This data suggests that AP-1 binding is required for responsiveness of this chronic TGFb inducible enhancer in melanoma.

Macrophages preferentially phagocytose TIE:EGFP expressing melanoma cells
We identified TIE:EGFP + macrophages in TIE:EGFP;Tg(mitfa:BRAF V600E );p53 -/-;mitfa -/-;MCR:MCS melanomas that express mitfa and sox10, suggesting recent phagocytosis of melanoma cells (Figure 3A).There are two potential models for why macrophages are TIE:EGFP + : (1) macrophages express TIE:EGFP themselves, or (2) macrophages are engulfing TIE:EGFP + cells.To test these hypotheses, we crossed TIE:EGFP;Tg(mitfa:BRAF V600E );p53 -/-;mitfa -/-zebrafish to a mpeg:mCherry reporter line that labels macrophages with mCherry (Ellett et al., 2011).These embryos were injected with MCR:BRAF V600E , 2x U6:p53/Tyr gRNA mitfa:Cas9, and mitfa:BFP to generate pigmentless, BFP + melanomas.As the TIE:EGFP reporter is cytoplasmic, if mpeg:mCherry + macrophages are endogenously expressing TIE:EGFP, we would expect the entire macrophage to appear yellow via imaging.Confocal imaging of these tumors showed that the majority of a TIE:EGFP + region of the tumor is composed of clustered TIE:EGFP + macrophages.Within these regions, we observed that a subset of macrophages contain only puncta of TIE:EGFP signal, suggesting phagocytosis of TIE:EGFP + cells, while a separate subset are entirely yellow (suggesting endogenous reporter expression; Figure 3B and Figure 3figure supplement 1A).In 10 out of 13 tumors analyzed by confocal microscopy, about 60-80% of TIE:EGFP + macrophages contained TIE:EGFP fragments, while the remaining TIE:EGFP + macrophages were diffusely TIE:EGFP + .In the remaining three tumors, about 30% of TIE:EGFP + macrophages contained TIE:EGFP fragments, while the remaining were diffusely TIE:EGFP + .Therefore, in most tumors (with rare exceptions), the majority of TIE:EGFP + macrophages are EGFP + because they phagocytosed a TIE:EGFP + cell.Indeed, we observed macrophages actively phagocytosing TIE:EGFP + cells (Figure 3    we observed that in TIE:EGFP + patches, macrophages were engaged closely with a cluster of TIE:EGFP + ;mitfa:BFP + tumor cells, likely in the process of phagocytosis (Figure 3B and Figure 3figure supplement 1B).Together, this data reveals that a large proportion of macrophages engulf a TIE:EGFP + cell population that includes melanoma cells.
To determine if macrophages are preferentially phagocytosing TIE:EGFP + melanoma cells, three TIE:EGFP + ;mpeg:mCherry + ;mitfa:BFP + melanoma were excised, digested, and processed for flow cytometry analysis (Figure 3C-D).Viable cells were separated into TIE:EGFP -and TIE:EGFP + (Figure 3D, far left).In all three tumors, less than 1% of sorted cells were TIE:EGFP + , indicating this TGFb responsive population is rare.TIE:EGFP -and TIE:EGFP + (Figure 3D, middle) cells were plotted relative to mpeg:mCherry and mitfa:BFP.Q2 represents the total percentage of TIE:EGFP -or TIE:EGFP + cells that were melanoma cells phagocytosed by macrophages.The percentage given in Q2 is plotted for several tumors in Figure 3D (far right).Macrophages phagocytose a significantly higher percentage of TIE:EGFP + melanoma cells compared to TIE:EGFP -melanoma cells.This was confirmed by qPCR, which showed that mitfa and sox10 expression is enriched in TIE:EGFP + macrophages compared to TIE:EGFP -macrophages (Figure 3-figure supplement 2).Altogether, scRNA-seq, confocal imaging and flow cytometry analyses demonstrate that macrophages preferentially phagocytose TIE:EGFP + melanoma cells compared to TIE:EGFP -melanoma cells.

SATB2 expression leads to early TGFb activation
The chromatin remodeler SATB2 is amplified in 4-8% of melanoma patients and its expression correlates with patient outcome.Our lab has previously shown that SATB2 overexpression leads to accelerated melanoma onset, produces more aggressive, metastatic tumors, and induces a TGFb signature (Fazio et al., 2021).SATB2 was overexpressed under the mitfa promoter in the MiniCoopR backbone (MCR:SATB2) in TIE:EGFP;Tg(mitfa:BRAF V600E );p53 -/-;mitfa -/-zebrafish.Of the fish with SATB2 melanomas (n=27), 68% turned on TIE:EGFP in tumors, and its signal was expressed throughout the tumor volume (Figure 4A).To understand the transcriptional differences between TIE:EGFP + and TIE:EGFP -melanoma cells, we sequenced the transcriptome of single cells from an MCR:SATB2 expressing melanoma at 30 wpf using SORT-seq (tumor used shown in Figure 4A, top).We identified most cells as melanoma cells with mitfa and/or sox10 expression, and again an mpeg1.1 expressing TIE:EGFP + macrophage population (Figure 4B and Figure 4-figure supplement 1A).
To determine if there is a change in state of the macrophages that express TIE:EGFP (either endogenously or via phagocytosis), we subset macrophages from the SATB2 expressing melanoma and separated macrophages that were TIE:EGFP + or TIE:EGFP -based on flow cytometry intensity.TGFb is known to convert macrophages from a pro-inflammatory M1-like to an anti-inflammatory M2-like state which can be characterized by gene expression.In the zebrafish, these include M1 markers acod1, tnfa, csf3a/b, and socs3b, as well as M2 markers mrc1b, vegfaa, alox5ap, and marco (Viola et al., 2019;Wu et al., 2015;Jeannin et al., 2018;Wilson, 2014;Martinez and Gordon, 2014;Hwang et al., 2020;Ye et al., 2021;Georgoudaki et al., 2016).We found that although TIE:EGFP + macrophages are not clearly polarized, they express a combination of M1-like and M2-like marker genes (Figure 4-figure supplement 1B).To understand the transcriptional differences between TIE:EGFP + and TIE:EGFP -melanoma cells, SATB2 expressing melanoma cells were divided into TIE:EGFP -, TIE:EGFP low , and TIE:EGFP high cells based on flow cytometry intensity.These MCR:SATB2 results confirmed what was observed in the TIE:EGFP + MCR:MCS tumors.We found that in TIE:EGFP high melanoma cells, chronic extracellular matrix TGFb target genes were up-regulated and the top down-regulated pathways by GSEA again were interferon alpha and gamma (Figure 4C and Figure 4-figure supplement 1C).Downregulation of interferon suggests that the TGFb responsive melanoma cells likely evade adaptive immune responses, such as interferon-mediated antigen presentation to CD8 + T cells.Finally, 40% of the MCR:SATB2 fish with melanomas (n=27) had TIE:EGFP expressed in early melanomas.These lesions are mitfa:mCherry high, indicating a proliferation of melanoma cells, but do not yet extend off the body plane.Meanwhile, 0% of MCR:MCS fish with melanomas (n=56) had TIE:EGFP + early melanomas (Figure 4D).This shows that overexpression of SATB2 leads to acceleration of TGFb response in melanoma and suggests patients with this amplification may be more susceptible to TGFb inhibitors.

Discussion
Here, we identified an inducible and specific TGFb enhancer reporter and visualized TGFb response throughout melanomagenesis in zebrafish.TIE:EGFP is off in early melanoma but is expressed in most advanced melanomas, and remains on, reading out chronic TGFb signaling.These TGFb responsive melanoma cells down-regulate interferon, up-regulate a series of novel chronic TGFb target genes, and are preferentially phagocytosed by macrophages.This work identifies a TGFb induced immune response and novel biomarkers of chronic TGFb signaling in melanoma.
We have shown that our reporter, generated from human ChIP-seq data, is able to read out chronic TGFb signaling.Most literature on developmental signaling focuses on acute signaling with stimulus for several hours in vitro.There are multiple explanations for why TIE:EGFP is a chronic TGFb reporter and once turned on, remains activated.Once the TIE:EGFP reporter is activated, it may remain active because chromatin is held in an open state by epigenetic modifications or AP-1 factors.We showed that AP-1 factors are necessary for TGFb induction of our TIE reporter and luciferase assays indicated that loss of AP-1 motifs eliminates basal levels of reporter transcription.AP-1 factors may be holding TIE chromatin open, therefore potentiating its ability to drive downstream reporter expression.Simultaneously, AP-1 factors likely hold chromatin open for other TGFb target genes to potentiate the TGFb response.This may allow for a rapid signaling response upon TGFb activation and suggests that AP-1 inhibitors could disrupt TGFb induction.
The TIE:EGFP scRNA-seq data indicates that melanoma cells responding to TGFb for several weeks see down-regulation of acute TGFb target genes.They exhibit up-regulation of 29 chronic which often appears as fragments within macrophages.A macrophage that expresses the TIE:EGFP endogenously would express EGFP throughout the entire cell, rather than in fragments.Cyan indicates a TIE:EGFP + melanoma cell.When phagocytosed by macrophages, TIE:EGFP + melanoma cells appear white, which are indicated within in the white boxes.(C) Representative TIE:EGFP + ;mpeg:mCherry + ;mitfa:BFP + melanoma used for flow analysis of macrophages.Scale bars indicate 1000 µm.The EGFP + region adjacent to the tumor is endogenous TIE:EGFP + expression of the brain.(D) (Far left) Viable cells were separated into TIE:EGFP -and TIE:EGFP + .(Middle) FACS plots showing TIE:EGFP -and TIE:EGFP + cells relative to mpeg:mCherry and mitfa:BFP.Q1 in the TIE:EGFP -plot represents macrophages that have not phagocytosed any melanoma cells.Q2 represents macrophages that have phagocytosed TIE:EGFP -melanoma cells.Q1 in the TIE:EGFP + plot represents macrophages that have not phagocytosed melanoma cells, but rather express the TIE:EGFP reporter endogenously or phagocytosed a TIE:EGFP + non-melanoma cell.Q2 represents macrophages that have phagocytosed TIE:EGFP + melanoma cells.(Far right) Q2 of both plots are graphed to represent the percentage of all TIE:EGFP -or TIE:EGFP + live cells that are melanoma cells phagocytosed by macrophages.Two-tailed unpaired Welch's t-test was used to calculate significance.n=3 fish with two technical replicates each.Illustrated fish diagram in (C) created with BioRender.com,and published using a CC BY-NC-ND license with permission.
© 2024, BioRender Inc. Figure 3 was created using BioRender, and is published under a CC BY-NC-ND license.Further reproductions must adhere to the terms of this license.
The online version of this article includes the following figure supplement(s) for figure 3:   TGFb target genes that are more representative of the downstream phenotypes imposed by TGFb signal, such as extracellular matrix genes.Based on the literature, these targets degrade extracellular matrix, promoting migration (Verrecchia et al., 2001).Some of the chronic TGFb targets were identified in one of the few reports of long-term TGFb signaling in human mammary epithelial cells for 12 or 24 days (Figures 2C and 4C).Prolonged TGFb treatment was found to stabilize EMT, stem cell state, and drug resistance in breast cancer cells (Katsuno et al., 2019).According to TCGA data in cBioPortal, a subset of melanoma patients exhibit up-regulation of these genes (Cerami et al., 2012;Gao et al., 2013).In the future, this signature could be used as biomarkers to identify patients with chronic TGFb signaling and these patients may benefit from treatment with TGFb inhibitors.
Overexpression of SATB2 resulted in TIE:EGFP expression in early melanoma lesions, which was not observed in controls.Cancer cells are thought to circumvent the tumor suppressive effects of TGFb via mutations or epigenetic modifications (Derynck et al., 2021;Kim et al., 2021;Massagué et al., 2000;Zhang et al., 2017;Seoane and Gomis, 2017;Suriyamurthy et al., 2019;Miyazawa and Miyazono, 2017;Tang et al., 2018).It is possible that melanomas up-regulating the epigenetic regulator SATB2 may overcome the tumor-suppressive effects of TGFb signaling earlier in tumor development, leading to more aggressive and invasive melanomas (Fazio et al., 2021).Investigating the activation of TGFb signaling in the context of different patient mutations may provide insight into who may benefit from TGFb inhibitors, allowing for the identification of biomarkers of TGFb inhibitor response.It is expected that aggressive melanoma subtypes like SATB2, which activate TGFb very early in melanoma development, would be most responsive to early intervention with TGFb inhibitors.
TIE:EGFP expression often occurs in patches throughout the tumor.In the tumor pictured in Figure 3C, there is general co-localization of mpeg:mCherry and TIE:EGFP signal indicating that macrophages may cluster in TGFb positive regions of the tumor.Using confocal imaging, we confirmed that TIE:EGFP signal is most often found in macrophages clustered in these zones.The TIE:EGFP reporter appears in some macrophages as a diffuse, cytoplasmic signal indicative of endogenous TGFb signaling.However in the majority of EGFP + macrophages, the reporter appears as fragments (sometimes even in endosome-like structures), indicating that many macrophages are TIE:EGFP + because they phagocytosed a TIE:EGFP + cell.We occasionally captured through confocal imaging macrophages in contact with TIE:EGFP + melanoma cells, suggesting that macrophages phagocytose these tumor cell populations (Figure 3-figure supplement 1).Indeed, our scRNA-seq data identified TIE:EGFP + macrophage subclusters that express mitfa and/or sox10, providing additional evidence for phagocytosis of melanoma cells.Using flow cytometry analysis, we showed that macrophages preferentially phagocytose TIE:EGFP + melanoma cells.The regionality of TGFb response could occur due to gradients in the TGFb morphogen, possibly induced by TGFb signaling within tumor cells in particular regions of the tumor.As mentioned above, TGFb can act as a chemoattractant for macrophages and induces an anti-inflammatory M2-like state.Once macrophages arrive to the TGFb region, they may phagocytose TIE:EGFP + tumor cells, thus explaining the presence of EGFP + fragments within macrophages.This data suggests that local TGFb signaling within the tumor may influence macrophage localization and phagocytosis, likely leading to changes in their behavior.
One factor we identified in our single cell RNA-seq data that may mediate this interaction is serpine1.Serpine1 mRNA was differentially upregulated in TIE:EGFP + melanoma cells, and its receptor, lrp1ab was expressed on TIE:EGFP + macrophages (data not shown).SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), has been shown to promote cancer cell invasiveness and macrophage SORT-seq of SATB2 expressing tumor in (A).Inset shows expression of these genes in the macrophage cluster.(C) Dotplot depicting extracellular matrix TGFb target gene expression in TIE:EGFP high , TIE:EGFP low , and TIE:EGFP -SATB2 expressing melanoma cells.Genes shown in red are genes that are also upregulated in control MCR:MCS tumors.(D) Early initiating melanoma (arrowhead) overexpressing MCR:SATB2 in TIE:EGFP;Tg(mitfa:BRAF V600E );p53 - /-;mitfa -/-zebrafish.Representative image chosen.40% of MCR:SATB2 early melanomas (n=27) express TIE:EGFP, compared to 0% of MCR:MCS early melanomas (n=56) (quantification on right).Illustrated fish diagrams in (A, D) created with BioRender.com,and published using a CC BY-NC-ND license with permission.
© 2024, BioRender Inc. Figure 4 was created using BioRender, and is published under a CC BY-NC-ND license.Further reproductions must adhere to the terms of this license.
The online version of this article includes the following figure supplement(s) for figure 4:  recruitment in an esophageal squamous cell carcinoma model (Sakamoto et al., 2021).TIE:EGFP high macrophages differentially express mmp14b and the cysteine protease legumain, both of which have been described to promote TGFb bioavailability (Bai et al., 2019;Sounni et al., 2010;Robertson and Rifkin, 2016).Together, these preliminary transcriptional data may indicate a mechanism by which macrophages are recruited to TIE:EGFP + regions by PAI-1 gradients.Recruited macrophages may phagocytose TIE:EGFP + tumor cells and then go on to amplify the pool of active, bioavailable TGFb through the activity of enzymes such as mmp14b and lgmn.Such a model would explain the local clustering of macrophages in TGFb-responding regions of the tumor, where a subset phagocytoses TIE:EGFP + melanoma and non-melanoma cells and others experience endogenous TGFb signaling (likely from local paracrine signaling).
Single cell RNA-seq data from the MCR:SATB2 melanoma suggests that TIE:EGFP + macrophages (which are either TGFb responsive themselves or had phagocytosed a TGFb responding cell) are not clearly polarized, expressing transcriptional markers of both M1 and M2 states.This may indicate that a transition is occurring from the M1-like to the M2-like phenotype.In our model, macrophages are attracted to TGFb expressing regions of the melanoma, and while phagocytosing in the vicinity of TGFb cytokines, begin to transition to an M2 state.M2 macrophages are known to be anti-inflammatory, immunosuppressive, and pro-angiogenic.Preliminary evidence comparing the transcriptional signatures of macrophages in the SATB2 expressing tumor indicates that TIE:EGFP + macrophages have higher expression of cholesterol and fatty acid genes (i.e.abca1a, npc2) as well as apoptotic genes (i.e.casp3b, caspa) compared to TIE:EGFP -macrophages.This indicates that intrinsic TGFb signaling or phagocytosis of TIE:EGFP + cells may induce a stress phenotype within macrophages, resulting in death.In this case, death of macrophages over time would allow the tumor cells to evade phagocytosis.This, in conjunction with down-regulation of interferon by TIE:EGFP + melanoma cells (as seen in MCR:MCS and MCR:SATB2 tumors), may lead to immune inactivation within the melanoma.Moreover, the interferon target gene, β-2-microglobulin ( b2m) is one of the top downregulated genes in TIE:EGFP expressing melanoma cells (Figure 2-figure supplement 1B).Loss of B2M, part of the MHC Class I molecules, is a known mechanism of immunotherapy resistance (Alavi et al., 2018).Together, this data suggests that activation of TGFb in melanoma is immunosuppressive, potentially by way of interferon modulation and effects on macrophage behavior and supports the need for more work on combination TGFb and immune checkpoint inhibitors.

Zebrafish work
All zebrafish (Danio rerio) work was performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Animal research protocols were approved by the Institutional Animal Care and Use Committee of Boston Children's Hospital, Protocol #20-10-4254R.All zebrafish work operated according to the guidelines of the Institutional Animal Care and Use Committee of Boston Children's Hospital.

Flow analysis
Tumors were excised and dissociated for 30 min with occasional chopping using 0.075 mg/mL Liberase (Sigma #5401119001) in DMEM (Gibco #11965-092) with 1% Penstrep (Corning #30-002 CI).Casper zebrafish skin was used to exclude autofluorescent cells (White et al., 2008).Mpeg:mCherry + and ubi:EGFP + zebrafish skin was used to set the gates for mCherry and EGFP intensity, respectively.A mitfa:BFP + patch of skin as well as flk:BFP + skin were used to set the gates for BFP intensity.Dissociated samples were filtered through a 40 µm filter and resuspended in FACS buffer (PBS/10%FBS/1% Penstrep) before filtering through a FACS tube (Corning #352235).DRAQ7 was used as a live/ dead marker (Abcam #ab109202, Cambridge, UK).Cells were analyzed using a BD FACS Aria II 5 Laser System.Two technical replicates of 1 million cells were sorted from each tumor, for a total of 2 million cells per tumor.Data was processed using FlowJo version 10.8.1 (RRID:SCR_008520).Debris, doublets, and autofluorescent cells were removed from the analysis and viable cells were separated into TIE:EGFP -and TIE:EGFP + .

Luciferase assays
Firefly luciferase reporter constructs (pGL4.24)were created by cloning the full and mutated TGFb enhancers upstream of the minimal promoter using BglII and XhoI sites (see Figure 2-figure supplement 2 for sequences).A375 cells were plated in opaque-walled 96-well plates (Thermo Fisher #136101) and approximately 5000 cells were co-transfected with 100 ng firefly and 10 ng Renilla luciferase plasmids using Lipofectamine 3000 (Invitrogen #L3000008).After 48 hr cells were serum starved for 2 hr and treated with 10 ng/mL TGFB1 or 4 mM HCl containing 1 mg/mL BSA vehicle control for an additional hr.Firefly and Renilla luciferase were then measured using the Dual-Glo Luciferase Assay (Promega #E2920, Madison, WI, USA) according to the manufacturer's instructions.Luminescence was read on a Synergy Neo plate reader and the ratio of firefly to Renilla luminescence was calculated.Empty firefly luciferase vector was used as a negative control and Renilla luciferase was used as control for transfection efficiency.Experiments were performed in biological triplicate with three technical replicates each.qPCR Two TIE:EGFP;mpeg:mCherry;Tg(mitfa:BRAF V600E );p53 -/-;mitfa:BFP tumors were dissociated for 30 min with occasional chopping using 0.075 mg/mL Liberase (Sigma #5401119001) in DMEM/F12 (Gibco # 11580546), and the reaction was inactivated by adding a solution of 15% FBS in DMEM/F12.Dissociated samples were filtered through a 40 µm filter and resuspended in FACS buffer (PBS/5% BSA).Casper zebrafish skin was used to exclude autofluorescent cells, and mpeg:mCherry + zebrafish skin was used to set the mCherry gate.SYTOX red dead cell stain was used as a live/dead marker (Invitrogen #S34859).Cells were sorted into FACS buffer in two separate tubes: mpeg:mCherry + TIE:EGFP + and mpeg:m-Cherry + TIE:EGFP -.RNA was extracted from cells sorted by FACS using the Direct-zol RNA MicroPrep Kit (Zymo Research #2060), and reverse transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen #11754-050).qPCR was performed using SYBR Green qPCR Mix (ThermoFisher # 4309155), and samples were run using the QuantStudio 6 Flex system (ThermoFisher).Mitfa primer sequences (FP: 5' CTAC GACA GCCC AAAC AAGG , RP: 5 ' GCCA TTGT CATG TTCG TCCA ).Sox10 primer sequences (FP: 5 'ACGC TACA GGTC AGAG T CAC, RP: ATGT TGGC CATC ACGT CATG ).Data was analyzed using the delta delta Ct method.Ct values were normalized to those of housekeeping gene, b-actin.B-actin primer sequences (FP: CGAG CAGG AGAT GGGA ACC, RP: CAAC GGAA ACGC TCAT TGC).

Statistics
To calculate significance of electroporation and flow analysis assays, a two-tailed unpaired t-test with Welch's correction was performed using GraphPad Prism version 9.0.2 (RRID:SCR_002798) for Mac (GraphPad Software, San Diego, California USA, https://www.graphpad.com).To calculate significance of luciferase assays, a 2-way multiple comparison ANOVA test was performed using GraphPad Prism version 9.0.2 for Mac.For the qPCR experiment in Figure 3-figure supplement 2, 2-tailed unpaired t-tests was performed using GraphPad Prism for Mac.

Figure supplement 2 .
Figure supplement 2. Treatment with human recombinant TGFB1 activates the TGFb pathway in human melanoma cells.

Figure 1 continuedFigure 2 .
Figure1 continued while macrophages were identified as mpeg1.1 and marco positive.(B) Dotplot depicting TGFb immediate-early target gene expression in TIE:EGFP high , TIE:EGFP low , and TIE:EGFP -melanoma cells.Melanoma cells can be segregated into TIE:EGFP high vs.TIE:EGFP low based on EGFP intensity during sorting.(C) Dotplot depicting extracellular matrix TGFb target gene expression in TIE:EGFP high , TIE:EGFP low , and TIE:EGFP -melanoma cells.(D) HOMER motif analysis of regulatory regions bound by SMAD2/3 upon stimulation in A375 cells.(E) Heatmap showing binding of JUNB and ATF3 pre-stimulus at 12,000 SMAD2/3-responsive elements in A375.(F) (Top) IGV plot of H3K27ac, SMAD2/3, ATF3 and JUNB ChIP-seq +/-TGFB1 stimulus at the TGFbinduced enhancer.Inset depicts sequence under SMAD2/3 ChIP-seq peak and highlights AP-1 (orange) and SMAD2/3 (red) binding sites.(Bottom) Firefly luciferase luminescence of full TIE reporter or reporter lacking AP-1 or SMAD2/3 sites.Normalized to Renilla transfection control.Experiment performed three times with three technical replicates each.A two-way multiple comparison ANOVA was used to calculate significance.The online version of this article includes the following source data and figure supplement(s) for figure 2:

Figure supplement 2 .
Figure supplement 2. Mutated TGFb inducible enhancer sequences used for luciferase assays in Figure 2F.

Figure
Figure supplement 2-source data 1.Editable version of Figure 2-figure supplement 2.
Figure 4 continued